Cytological Evaluation

Abstract

The fact that the human body is composed of cells was demonstrated by Dr. Hooke in 1665, and modern cytology was initiated by Dr. Papanicolaou in 1928. In 1943, Papanicolaou published “Diagnosis of Uterine Cancer by Vaginal Cytology,” which is the basis of current cytodiagnostics. There are several types of materials used in cytological diagnosis, each with different methods of specimen preparation and determination. The advantages of some specimen collection methods are that they are less invasive to the body and provide quick determination results. However, correct results cannot be obtained unless the specimen is prepared correctly. In recent years, various methods have been developed with the advancement of imaging tests, the spread of genetic testing, and the spread of artificial intelligence (AI). Liquid-based cytology (LBC) has become widespread, especially in gynecology. It has also become possible to test for human papillomavirus (HPV), which is closely related to cervical cancer, using the same materials. An automated screening system based on a database of morphological characteristics of atypical/tumor cells has also been developed.

1. Introduction

Cytodiagnostics began in 1928 when modern cytology was commenced by Dr. Papanicolaou and was initially recognized for its usefulness as a diagnostic method for cervical cancer (see Figure 1) [1]. The examination of smears by Dr. Papanicolaou is one of the most important historical achievements in screening methods for the prevention of disease and cancer. It has been used as a screening test for uterine cancer since 1950. The test is straightforward and practical from a technical perspective and utilizes the simple scientific observation that malignant cells have abnormal nuclear morphology that can be distinguished from benign cells [2, 3, 4]. The International Academy of Gynecological Cytology (IAC), which was founded in 1957 and originated from the Japan Gynecological Cytology Discussion Group, was established in Japan in response to the International Academy of Gynecological Cytology (IAGC).

Figure 1.

Gynecological cytology by Dr. Papanicolaou. (a) Normal. (b) Inflammation. (c) Carcinoma. (a) a moderate number of inflammatory cells are seen in the background, and superficial-to-medium squamous epithelium is present. The patient is presumed to be an adult female. (b) A strong inflammatory background is present. (c) The cells differ in enlargement from (a) and (b), but have nuclei that are large and show shape irregularities. Quoted from reference [1], partially modified. George N. Papanicolaou. M.D, New Cancer Diagnosis, The Third Race Betterment Conference, Battle Greek. Michigan. January 2-6. 1928. And published in the proceedings of the Conference the same year.

The Japanese Society of Clinical Cytology is a professional society that promotes academic research in clinical cytology and cytodiagnostics and the application of its results to practical clinical practice. Its origins date back to 1961.

Later, in line with advances in other fields of science, the IAC was renamed the International Academy of Cytology in 1961. In 1962, it was joined in Japan with the Tokyo Cytodiagnosis Study Group and others to be known as the Japanese Society of Clinical Cytology.

Since the early 2000s, the academic content of this society has covered numerous fields, including gynecology, surgery, internal medicine, pathology, dentistry, and especially cancer diagnosis. Notably, it is valuable as a means of cancer screening and is used for cervical, uterine, lung, and breast cancer screening. Among these, with its long history, cervical cancer screening has achieved significant results for reducing disease mortality. The Papanicolaou classification was used as a criterion (see Table 1). Cells are classified according to their morphological changes from benign (normal or near-normal cells) to malignant (cancerous cells). The Papanicolaou classification, which has five levels from Class I (no abnormality) to Class V (malignant), has been widely used in gynecological cytology because it is easy to compare with histological diagnosis and is common and convenient for other organs.

Class	Comments
I	No abnormal cells
II	Abnormal or abnormal cells present, but no malignant findings
III	Abnormal cells suspected of being malignant are found, but they cannot be determined to be malignant
IIIb	Abnormal cells strongly suspected of being malignant are found, but cannot be determined as malignant
IV	Abnormal cells with strong suspicion of malignancy are observed
V	Recognize cells that can be determined to be malignant

Table 1.

Papanicolaou’s classification from the Japanese Society of Clinical Cytology based on gynecology.

Papanicolaou classifications I–V were established in cytology in Japan as the main classification method. Together with the Japanese grouping of the gastrointestinal tract, it supports an easy understanding of the stages of cancer. This classification was believed to be universal and unchanging, and thus, it was perplexing that “overseas, papers written using the Papanicolaou classification are not accepted.” Twenty years ago, the Washington Post reported on the low rate of positive cytological diagnoses in the USA using the Papanicolaou classification, and this led to the establishment of the Bethesda system (see Table 2) [5, 6, 7, 8, 9, 10, 11].

Specimen type

Indicate conventional smear (Pap smear), liquid-based preparation (Pap test) vs other

Specimen adequacy

• Satisfactory for evaluation (describe presence or absence of endocervical/transformation zone component and any other quality indicators, e.g., partially obscuring blood, inflammation, etc.)

• Unsatisfactory for evaluation (specify reason)

○ Specimen rejected/not processed (specify reason)

○ Specimen processed and examined, but unsatisfactory for evaluation of epithelial abnormality because of (specify reason)

General categorization (optional)

• Negative for intraepithelial lesion or malignancy

• Other: see Interpretation/Result (e.g., endometrial cells in a woman aged ≥ 45 years)

• Epithelial cell abnormality: see Interpretation/Result (specify “squamous” or “glandular,” as appropriate)

Interpretation/result

Negative for Intraepithelial Lesion or Malignancy

(When there is no cellular evidence of neoplasia, state this in the General Categorization above and/or in the Interpretation/Result section of the report—whether or not there are organisms or other non-neoplastic findings)

Non-Neoplastic Findings (optional to report)

Non-neoplastic cellular variations

○ Squamous metaplasia

○ Keratotic changes

○ Tubal metaplasia

○ Atrophy

○ Pregnancy-associated changes

Reactive cellular changes associated with:

■ Inflammation (includes typical repair)

○ Lymphocytic (follicular) cervicitis

■ Radiation

■ Intrauterine contraceptive device (IUD)

Glandular cells status posthysterectomy

Organisms

Trichomonas vaginalis

Fungal organisms morphologically consistent with Candida spp.

Shift in flora suggestive of bacterial vaginosis

Bacteria morphologically consistent with Actinomyces spp.

Cellular changes consistent with herpes simplex virus

Cellular changes consistent with cytomegalovirus

Other

○ Endometrial cells (in a woman aged ≥ 45 years)

(Also specify if “negative for squamous intraepithelial lesion”)

Epithelial Cell Abnormalities

Squamous Cell

• Atypical squamous cells

○ Of undetermined significance (ASC-US)

○ Cannot exclude HSIL (ASC-H)

• Low-grade squamous intraepithelial lesion (LSIL)

(Encompassing: HPV/mild dysplasia/CIN-1)

• High-grade squamous intraepithelial lesion (HSIL)

(Encompassing: moderate and severe dysplasia, CIS; CIN-2 and CIN-3)

○ With features suspicious for invasion (if invasion is suspected)

• Squamous cell carcinoma

Glandular cell

• Atypical

○ Endocervical cells (NOS or specify in comments)

○ Endometrial cells (NOS or specify in comments)

○ Glandular cells (NOS or specify in comments)

• Atypical

○ Endocervical cells, favor neoplastic

○ Glandular cells, favor neoplastic

• Endocervical adenocarcinoma in situ

• Adenocarcinoma

○ Endocervical

○ Endometrial

○ Extrauterine

○ Not otherwise specified (NOS)

Other Malignant Neoplasms (specify)

Adjunctive testing

Provide a brief description of the test method(s) and report the result so that it is easily understood by the clinician

Computer-assisted interpretation of cervical cytology

If case examined by an automated device, specify the device and result

Educational notes and comments appended to cytology reports (optional)

Suggestions should be concise and consistent with clinical follow-up guidelines published by professional organizations (references to relevant publications may be included)

Abbreviation: CIN, cervical intraepithelial neoplasia; CIS, carcinoma in situ; HPV, human papillomavirus; NOS, not otherwise specified; Pap, Papanicolaou.

Table 2.

The 2014 Bethesda system.

Source: Reprinted from [5].

Therefore, why the positive diagnosis rate was so low with the Papanicolaou classification became a question. Various theories exist, including that the specimens were of poor quality, there was an insufficient number of cells smeared, and fixation or staining was not performed properly. Therefore, emphasis was placed on whether enough cells had been collected, whether fixation was adequate, and whether the specimens had been prepared appropriately.

Cytology results are ultimately a judgment based on the individual cytological image obtained. A proper judgment cannot be made if an insufficient amount of sample was collected, thus demonstrating that a necessary quantity must be obtained. This suggests that without proper sampling, accurate determination is not possible. The following section describes the types of collection material and critical points and precautions for specimen preparation. This is very important for a correct assessment.

It has been revised in various ways, and now, some 60 years later, current gynecological cytology is undergoing further changes. Liquefied cytology, which was introduced in Japan later than in Western countries, is becoming more widespread. It is now possible to perform human papillomavirus (HPV) testing on samples taken at the same time [12, 13, 14, 15]. This is the time when the efforts for the early detection and treatment of cervical cancer are relevant in terms of cytological diagnosis tests. Furthermore, automatic screening has been initiated.

Cytological tests are now being actively considered not only for cell morphology diagnosis, but also for the introduction of new technologies. These include molecular cytological research on genetic mutations during tumorigenesis and abnormal expression of oncogenes, automated cell diagnosis, and telecytology as part of telemedicine.

2. Materials for cytology determination and evaluation and notes on specimen preparation

Cytodiagnosis is a test that involves the observation of cell images taken from a patient to determine the benign/malignant nature of the cells obtained, among other things.

Cytodiagnosis can be performed by collecting cells in body fluids, such as sputum, urine, pleural fluid, or ascites, or through cell collection with a needle or rubbing with a brush. Depending on the method used to collect the cells, it is classified as exfoliative cytology, puncture aspiration cytology, abrasion cytology, or imprint cytology.

2.1 Exfoliative cytology

Cells spontaneously detach from tissue, often into liquid, such as sputum, urine, milk, pleural fluid, and ascites. The obtained cytogram is often accompanied by degeneration. Care must be taken to consider this part of the image when making a decision. However, there is little burden on the patient, such as pain.

2.2 Abrasion cytology

Cells are collected by rubbing the surface of the tissue in the cervix and bronchi. Cell images should be dried without ethanol fixation as soon as possible after the smear.

2.3 Imprint cytology

Cells are collected by rubbing tissue taken from biopsies or surgery onto a glass slide. As with abrasion cytology, fixation is required immediately after the smear. Care should also be taken not to spoil lymph nodes or undifferentiated tumors if they are suspected.

2.4 Puncture aspiration cytology

The needle test is used to collect lesions from areas under the skin, such as the mammary glands, thyroid gland, and lymph nodes. Fresh cells are obtained, and care must be taken to ensure that the smear and preparation of the specimen are performed as close as possible to recapitulate the situation in vivo. Introducing artifacts will prevent correct assessment and diagnosis.

Approximately 10–15 years ago, there was a debate in the Japanese domestic cytological society about the smear method for mammary gland puncture aspiration cytology. The author believes that the smear should be blown at an angle and fixed as it is (see Figure 2). At that time, there was an opinion within the academic society that lightly fitting two glass slides together was the correct approach. However, when a trace amount of liquid component is placed between the glass slides, it is technically impossible to lightly align them because of the surface tension, ruining the precious sample.

Figure 2.

Standard correct method of fine needle aspiration cytology. (excerpt from an educational video provided by AstraZeneca). (a) this is performed to confirm that the needle is definitely inside the tumor and is done with ultrasound guidance. When the needle tip enters the target site (inside the tumor) aspirate for about 1 minute. Keep the pistol primed (do not scatter cancer cells in the needle). Rotate the needle and gently move it back and forth to push the tumor cells into the needle. Insert, and always return suction when removing the needle. (b) Angle the tip of the needle about 40° and lightly push. Blow onto a glass slide. (c) Point the cut surface of the needle point slightly diagonally instead of straight down. When sprayed, the cells are dispersed almost evenly. (d) Spray the smeared slide glass as it is quickly fixed in alcohol. (e) When remove the alcohol, if it is smeared to the extent shown here, then it is generally okay. (f) Sometimes cells remain at the base of the needle. (g) If this occurs, cap the needle upside down on a slide glass and hit strongly.

The author had a bitter experience in understanding this, as shown in Figures 3 and 4. These are cases in which the smears are made by matching the glass and destroying the cells; thus, a definitive diagnosis cannot be made. At an academic conference in Japan, several researchers stated that a thick smear is not good because it does not allow the staining solution to enter the smear. For example, if a specimen is cancer, the tumor is a manifestation of a fast cell proliferation rate and high cell density. Adding an artifact to force the stain to cover the tumor in its entirety would destroy the structure of the finalized cell population and result in misdiagnosis. Thus, staining cytology specimens should not be mistaken for paintings.

Figure 3.

Fibroadenoma. (a) Laminated glass smear method. (b) Spray smear method. (a) Reported as class III because the cells collapsed due to rubbing together and could not be accurately determined. (b) The bilayered structure of myoepithelial and glandular cells is clear because the cells are not broken.

Figure 4.

Ductal carcinoma. (a) Laminated glass smear method. (b) Spray smear method. (c) Smashing smears. (d) Macroscopic image of slide specimen: (d-1) use of the spray smear method; (d-2) use of smashing smears. (a) the individual cells are stretched by the effects of rubbing together. (b, c) the cancer cells have not collapsed. The stacked structure is also preserved.

At symposiums and other meetings of academic societies in Japan, the results were not determined, and the tests were subsequently performed using the chosen approach of each institution. As a result, since around 2015, the rate of positive diagnosis has declined, especially for puncture aspiration cytology of the mammary gland. Guidelines for surgical and treatment methods also indicated tissue biopsy using a thick biopsy needle as the mainstream approach, while preoperative testing for hormone receptors, HER2 sensitivity, and Ki-67 index became the standard [16, 17, 18].

We hope that the time will come when puncture aspiration cytology will be actively utilized for the follow-up of benign lesions, such as fibroadenomas.

Pancreatic cancer is among the most lethal cancers worldwide because of the limited availability of techniques for the early detection of signs and symptoms. Ultrasound-guided pancreatic puncture aspiration is now also actively performed in some major hospitals [19, 20]. In some hospital facilities, cytological examinations and tissue biopsies are performed together. As the author noted [21], both tests have their own advantages and disadvantages. Unlike tissue biopsy, cytology allows specimens to be obtained from background findings. In the preparation of the tissue specimen, the background findings are the parts that are removed by roughing the paraffin block [21]. The presence of cystic or necrotic material in the specimen is a very important diagnostic finding (see Figure 5).

Figure 5.

Adenocarcinoma of the pancreas. (a) Cytology, class V. necrotic material was obtained in the background indicated by the red arrows (). (b) Histology, no malignancy. Only red blood cells and histiocytes were observed in the background. Source: Excerpted from ref. [21], partially modified.

2.5 Cell block specimens

Traditionally, cell block specimens have been used when fibrin precipitation has caused cells to adhere to each other and clump together, making smears on glass slides impossible. Sometimes, useful findings can be obtained when the clumped material is used for specimen preparation in the same way as tissue fragments [22, 23, 24]. Therefore, immunostaining has recently become increasingly necessary. The reasons for this are the emergence of cancers of unknown primary origin and the widespread use of antibodies for immunostaining.

More recently, when the only symptom is fluid accumulation in the body cavity, such as pleural or ascites fluid, this has been diagnosed as cancer on cytological examination. However, imaging findings of the surrounding organs are not abnormal. Therefore, cell block specimens are prepared from the remaining smear sediment; then, immunostaining is performed to deduce the primary tumor (see Figure 6). Based on this, an increasing number of centers are processing specimens with the aim of preparing cell blocks from the outset. Body cavity fluid, which is collected in large quantities, is useful because it tends to have surplus sediment.

Figure 6.

Microscopic findings for lung adenocarcinoma case. (a) Cytological findings (Papanicolaou). (b) Cell block findings (hematoxylin and eosin). (c) Cell block findings (immunohistochemistry for CEA). (d) Cell block findings (immunohistochemistry for ADC cocktail (TTF-1 and Napsin A)). Excerpt from ref. [24], partially modified.

Specifically, this process uses disposable tubes made of soft material, as shown in Figure 7. Samples are rotated faster than normal centrifugation (3000 rpm, 5 min); then, the supernatant is removed and fixing solution (15% neutral buffered formalin) is added. The sample is left to fix for approximately one day. The specimen is then cut with scissors as shown in the diagram, placed in an embedding cassette, and processed in an automatic embedding machine. Specimens are then prepared in the same way as normal tissue specimens. Ideally, during paraffin embedding, the sediment is picked up with tweezers from inside this tube and embedded directly into the embedding dish, but thin sections can also be made by embedding the entire Japanese tube. An alternative method is to use collodion. The use of sodium alginate has also been considered but should be avoided because it has been reported that sodium alginate may cause some antibodies to become unstained in rare cases.

Figure 7.

The cell block producing method. (a) Sample is fixed with 15% formalin solution in tube. (b) Cut the tip of the disposable tube using scissors. (c) The tip of a tube is placed in a cassette. Excerpt from ref. [24], partially modified.

3. Methods introduced in recent years

3.1 ThinPrep integrated imager system mirror inspection support system

In Japan, the cervical cancer screening uptake rate is lower than in Europe and the USA. In addition to improving the uptake rate, training cytologists for screening have become an issue [25, 26, 27]. In Europe and the USA, the introduction of liquid-based cytology (LBC) and automatic screening support devices with artificial intelligence (AI) has been progressing to support the high cervical cancer screening uptake rate. Such automatic screening support devices are expected to become widespread in Japan in the future. When one hears the term “automated screening support system,” one may wonder if it is safe. However, when a cytotechnologist performs the screening, it is undertaken by a human, which entails the risk of oversight and other issues. The concept of this system is to avoid overlooking high-grade squamous intraepithelial lesion (HSIL) lesions.

The system performs two ThinPrep smears for each case. One of them is stained using ThinPrep stain, a staining solution that differs from the usual Papanicolaou stain. The ThinPrep-stained specimens are then set in the machine in advance and read through, and 22 areas that need to be checked again are selected. These areas are checked with the cytologist using the special microscope shown in Figure 8 to make a final determination (see Figure 9).

Figure 8.

ThinPrep® integrated imager. Marketing authorization number; 13B1X10179001003.

Figure 9.

ThinPrep® imaging system workflow.

This machine (special microscope) shown in Figure 10 is available from Hologic. A comparison of actual cytologist specimens (M-microscopy) and I2-microscopy (machine) is also shown (see Table 3).

Figure 10.

Manual screening vs. integrated imager screening (I2 screening). (a) M screening: LSIL histology: HSIL/CIN3. (b) I2 screening: HSIL histology: HSIL/CIN3. (c) M screening: Lost histology: HSIL/CIN.

Table 3.

Comparison of M screening and I2 screening with Histology.

Agreement rate with tissue diagnosis: LSIL.

M screening: 95.0%.

I2 screening: 97.5%.

Agreement rate with tissue diagnosis: HSIL or higher.

M screening: 60.0%

I2 screening: 86.6%

26.6% up!

Notes)

M screening: Manual screening.

I2 screening: Integrated Imager screening.

Quoted from reference [26], partially modified. Materials from department of Pathology, Osaka Police Hospital and Hologic, Inc.

ThinPrep-stained specimens appear very dark compared with the normal Papanicolaou stains. However, many of the cells with some atypia have increased chromatin, so they are not likely to be observable by eye unless they are stained somewhat darkly.

The machine then identifies atypical cells at a certain percentage of sensitivity without tiring, unlike humans. In the near future, Japan’s aging population is expected to become a serious issue, and this and other factors will increase the need to rely on machines.

However, the author believes that this system would not be effective unless we can make an accurate judgment by looking at the cells presented by this special microscope. It may be meaningless unless the cytologist has the skills to make accurate judgments when presented with several fields of view.

Although Hologic’s system requires the preparation of LBC specimens, other companies have developed and marketed systems that allow the use of conventional smears. However, it is assumed that it is quite difficult to create a database based on the conventional method. The system is actually being used by a major testing company, and it is assumed that a considerable number of specimen images are being compiled into a database before it can be used in the current system.

3.2 Combination with HPV testing

HPV testing can now be performed on the same vial from which the LBC specimen is taken for the purpose of cytological diagnosis testing. HPV detection reagents are available from Qiagen, Roche, and BD [28, 29, 30]. This seems to be an excellent system for the early detection of dysplastic lesions and the eradication of cervical cancer from both the polymerase chain reaction data and the morphology on the cytogram. In addition, HPV vaccination, which had been on the decline because of adverse reactions, has been actively resumed in Japan.

4. Conclusion

A century has passed since Dr. Papanicolaou began his cytology research. Many cytotechnologists and cytology specialists have played a central role in performing various studies from all sides with the aim of achieving accurate diagnosis. As a result, various meaningful methods have been developed. However, some fields have declined discussions and have become divided and ambiguous. Furthermore, the emergence of AI may cause cytotechnologists to lose their jobs. However, in hindsight, cytology is being effectively used, which is possibly unique to the modern area. This is considered to be proof that cytological diagnosis is highly evaluated worldwide.

Conflict of interest

The author declares no conflict of interest.